Modulation of Wound Healing and Scar Formation by MG53Protein-mediated Cell Membrane Repair*□S

Received for publication, July 21, 2015, and in revised form, August 24, 2015 Published, JBC Papers in Press, August 25, 2015, DOI 10.1074/jbc.M115.680074

Haichang Li‡1,2, Pu Duann‡1, Pei-Hui Lin‡, Li Zhao‡§, Zhaobo Fan¶, Tao Tan‡§, Xinyu Zhou‡, Mingzhai Sun‡,Minghuan Fu¶, Matthew Orange�, Matthew Sermersheim‡, Hanley Ma‡**, Duofen He‡‡, Steven M. Steinberg‡,Robert Higgins‡, Hua Zhu‡, Elizabeth John§, Chunyu Zeng‡‡, Jianjun Guan¶, and Jianjie Ma‡§3

From the ‡Department of Surgery, Davis Heart and Lung Research Institute, ¶Department of Materials Science and Engineering,Ohio State University, Columbus, Ohio 43210, the §Division of Protein Therapeutics, TRIM-edicine Inc., Columbus, Ohio 43212, the�Department of Physical Education and Human Performance, Central Connecticut State University, New Britain, Connecticut06050, **Olentangy Liberty High School, Powell, Ohio 43065, and the ‡‡Department of Cardiology, Chongqing Institute ofCardiology, Daping Hospital, The Third Military Medical University, Chongqing 400042, China

Background: MG53 is a membrane repair gene whose role in wound healing has not been studied.Results: Topical administration of MG53 protein facilitates wound healing and reduces scar formation.Conclusion: This study establishes MG53 as facilitator of injury repair and inhibitor of myofibroblast differentiation duringwound healing.Significance: MG53 has therapeutic benefits in treating wounds and fibrotic diseases.

Cell membrane repair is an important aspect of physiology,and disruption of this process can result in pathophysiology in anumber of different tissues, including wound healing, chroniculcer and scarring. We have previously identified a novel tripar-tite motif family protein, MG53, as an essential component ofthe cell membrane repair machinery. Here we report the func-tional role of MG53 in the modulation of wound healing andscarring. Although MG53 is absent from keratinocytes andfibroblasts, remarkable defects in skin architecture and collagenoverproduction are observed in mg53�/� mice, and these ani-mals display delayed wound healing and abnormal scarring.Recombinant human MG53 (rhMG53) protein, encapsulated ina hydrogel formulation, facilitates wound healing and preventsscarring in rodent models of dermal injuries. An in vitro studyshows that rhMG53 protects against acute injury to keratino-cytes and facilitates the migration of fibroblasts in response toscratch wounding. During fibrotic remodeling, rhMG53 inter-feres with TGF-�-dependent activation of myofibroblast differ-entiation. The resulting down-regulation of � smooth muscleactin and extracellular matrix proteins contributes to reducedscarring. Overall, these studies establish a trifunctional role forMG53 as a facilitator of rapid injury repair, a mediator of cellmigration, and a modulator of myofibroblast differentiationduring wound healing. Targeting the functional interaction

between MG53 and TGF-� signaling may present a potentiallyeffective means for promoting scarless wound healing.

Wound care is a major challenge to public health, and theburden for wound treatment is growing because of an agingpopulation and a sharp rise in the incidence of diseases such asdiabetes and obesity that contribute to poor wound healing. Inthe United States, �6.5 million patients are affected by chronicwounds, and more than 72,000 amputations occur each year(1). Inadequate regenerative capacity during wound healingoften results in scar formation, caused mainly by the depositionof extracellular matrix (ECM)4 proteins (2–5). Although multi-ple factors may contribute to wound healing complications,compromised tissue repair capacity represents an underlyingcause for wounds that fail to heal. Development of therapeuticapproaches that facilitate scarless wound healing represents animportant area of biomedical and clinical research.

Several critical steps are involved in wound healing, includ-ing the initial inflammation caused by the acute death of cellswithin the wound area and release of pro-inflammatory factors(2– 4). Although recruitment of inflammatory cells to thewound environment is important for prevention of bacterialinfection, excessive inflammation is often associated with thedeath of cells and delayed wound healing. During the prolifer-ative stage of wound healing, the release of cytokines andgrowth factors, such as TGF-�, stimulates the migration ofkeratinocytes and fibroblasts toward the wound area to beginthe re-epithelialization and tissue rebuilding process (6, 7). Theproliferative phase also involves the conversion of fibroblastsinto myofibroblasts that secrete ECM proteins, which arerequired for closure of the wound. It is known that excessivesynthesis of ECM components because of uncontrolled differ-

* This work was supported, in whole or in part, by National Institutes of HealthGrants R01-AR061385 and R01-HL069000 (to J. M.). This work was also sup-ported by National Science Foundation grants 1006734 and 1160122 (toJ. G.) and the Ohio State University Lockwood Early Career DevelopmentAward (to P. L. and H. L.). J. M. and T. T. have an equity interest in TRIM-edicine, which develops MG53 for the treatment of human disease. Pat-ents on the use of MG53 are held by Rutgers University Robert Wood John-son Medical School.

□S This article contains supplemental Movie 1.1 Both authors contributed equally to this work.2 To whom correspondence may be addressed: Tel.: 614-292-3012; E-mail:

Haichang.Li@osumc.edu.3 To whom correspondence may be addressed: Tel.: 614-292-2636; E-mail:

Jianjie.Ma@osumc.edu.

4 The abbreviations used are: ECM, extracellular matrix; rh, recombinanthuman; �-SMA, � smooth muscle actin; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; FN, fibronectin.

crossmarkTHE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 290, NO. 40, pp. 24592–24603, October 2, 2015

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entiation of myofibroblasts can lead to pathologic scarringassociated with tissue injuries. Although many studies havebeen designed to target TGF-� signaling for the treatment ofwound healing, these attempts often faced a double-edgedsword. For example, blocking TGF-� signaling can reduce scar-ring but also compromise wound healing and other cellularfunctions (8 –11).

Cell membrane injury repair is an important aspect of nor-mal physiology, and its disruption can result in various pathol-ogies, including insufficient wound healing, chronic ulcers, andscarring (1–3, 12–15). We previously identified a novel tripar-tite motif family protein, MG53, as an essential component ofthe cell membrane repair machinery (14). MG53 functions invesicle trafficking and allows the nucleation of intracellular ves-icles at sites of membrane disruption. MG53 knockout mice(mg53�/�) exhibit defective membrane repair in striated mus-cle that leads to progressive skeletal myopathy and increasedvulnerability of cardiomyocytes to ischemia-reperfusion-in-duced injury (14, 16, 17). Moreover, the recombinant humanMG53 (rhMG53) protein can protect various cell types againstmembrane disruption when applied to the extracellular envi-ronment and ameliorate the pathology associated with muscu-lar dystrophy and lung injury (18, 19). We have also shown thatrhMG53 is effective in protection against myocardial infarction(20) and acute kidney injury (21).

In this study, we present evidence supporting the biologicalfunction of MG53 in the modulation of wound healing andscarring. First, MG53 is capable of nucleating the cell mem-brane repair machinery, therefore protecting from acute andstress-induced injuries to the epidermis. Such a fast membraneresealing response could reduce the overall inflammatoryresponses during the early phase of wounding and not onlyfacilitate early closure of the wound but also impact the fibroticremodeling during later stages of healing. Second, we foundthat MG53 present in the extracellular solution can facilitatemigration of fibroblast cells in response to scratch wounding,which is an important step in efficient wound healing. Third, wefound that MG53 can interfere with TGF-� signaling to controlthe differentiation process of fibroblasts into myofibroblasts.The latter is a major source of secretion of ECM proteinsand the main factor contributing to scarring. We show thattreatment of fibroblasts with rhMG53 leads to reduced expres-sion of �-SMA, collagen 1, and fibronectin, which may contrib-ute to the reduction of scarring during wound healing.

Experimental Procedures

Chemicals and Recombinant Human MG53 Protein—Allchemicals were obtained from Sigma-Aldrich (St. Louis, MO)unless stated otherwise. rhMG53 protein was purified fromEscherichia coli fermentation as described previously (18).rhMG53 was stored as lyophilized powder and dissolved insaline solution or encapsulated in hydrogel before use.

Animal Care and in Vivo Wound Healing Models—All ani-mal care and use followed National Institutes of Health guide-lines and Institutional Animal Care and Use Committeeapproval by Ohio State University. mg53�/� mice and wild-type littermates were bred and generated as described previ-ously (14). Sprague-Dawley rats (250 –300 g in weight) were

purchased from Charles River Laboratories. During surgicalprocedures, animals were anesthetized using isoflurane. Surgi-cal excision or incision wounds were created as detailed below.For pain management, all animals received drinking water con-taining ibuprofen (PrecisionDoseTM, 200 mg/liter in water) for2 days prior to and 5 more days post-surgery.

Excisional Wound Model—Dorsal hair was shaved usingelectric clippers, and the area was disinfected by application ofbetadine solution. Alcohol prep pads were then used to wipethe area clean. Two full-thickness dermal wounds (3 cmapart and 4 cm caudal to the scapulae) were created usingsterile 5-mm- (mice) or 6-mm-diameter (rats) biopsypunches (IntegraTM Miltex�) to expose the underlying dor-solateral skeletal muscle fascia. Hemostasis was achieved byeven compression with sterile gauze. Wounds were left open withno dressing, and then the mice immediately received a subcutane-ous injection of either 200 �l of saline (as a control) or rhMG53 (1mg/kg) and were treated daily for 3 successive days.

Rat Incision Wound Model—The hind limbs were shaved anddisinfected, and marks of 10-mm length were made to guide theincisional wound. Incisional wounds 10 mm long and 3 mmdeep were created on both limbs on the gastrocnemius musclewith a sharp blade (World Precision Instruments, Inc.).Wounds were treated subcutaneously with saline or rhMG53(0.2 mg/wound) in a similar fashion as described above. For thehydrogel experiment, a single dose of hydrogel or rhMG53/hydrogel (containing 0.2 mg of rhMG53) was applied topically.Wounds were left open with no dressing.

Rabbit Incision Wound Model—Four New Zealand rabbitswere used for this study. Three incisional wounds of 3 cm(length) � 1 cm (depth) were created on the back of each rabbit.Hydrogel or rhMG53/hydrogel (0.1 mg rhMG53/wound) wasapplied to the wound immediately after injury, and then theincision was closed with sutures. Of the 12 wounds generated,six received hydrogel and six received rhMG53/hydrogel.

Wound Analysis—A digital camera (Panasonic DMC-ZS3)was used to capture the wound with a metric ruler inside thefield of view to set the scale for measurements. The wound bedwas measured using digital calipers (CD-6”CSX, MitutoyoCorp.). Blinded measurement of wound size was performedusing ImageJ software. The perimeter of the wound was thentraced, and the wound area was recorded. The wound size onday 0 was set to 100 and, on each subsequent day, was reportedas a percentage of the initial wound size.

Histopathology and Immunohistochemistry—Skin and mus-cle samples dissected from experimental animals were fixed in10% formalin overnight at 4 °C. Skin samples were pinned toStyrofoam rafts to maintain morphology during the fixation.After fixing, samples were washed three times for 5 min with70% ethanol. Washed samples were processed and embeddedin paraffin. 4-�m-thick paraffin sections were cut and stainedwith H&E and Masson trichrome. Immunohistochemicalstaining of MG53 in skin tissues was performed using the Ven-tana Medical Systems Discovery XT automated immunos-tainer. Deparaffinization and antigen retrieval was performedusing cell conditioning solution (CC1, Ventana Medical Sys-tems). Rabbit polyclonal anti-MG53 antibody (14) was appliedand incubated at 37 °C for 1 h. Donkey anti-rabbit secondary

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antibody (Jackson ImmunoResearch Laboratories) was appliedand incubated for 1 h at 37 °C. The chromogenic detection kitDABMap (Ventana Medical Systems) was used to label the sec-ondary antibody. Slides were counterstained with hematoxylin,dehydrated, and xylene-cleared. The resulting slides showedMG53 protein stained brown and surrounding tissue stainedblue.

Hydroxyproline Assay—Hydroxyproline is a major compo-nent of collagen that helps stabilize the helical structure of col-lagen. Therefore, its levels can be used as an indicator of colla-gen content (22). The hydroxyproline contents from the backskin of WT and mg53�/� mice were extracted and measuredusing a hydroxyproline assay kit (Sigma-Aldrich) according tothe instructions of the manufacturer. The tissue hydroxypro-line content (expressed as micrograms per milligram of skintissue) was analyzed.

Western Blot Analysis and Antibodies—Protein lysates fromthe indicated tissue and cell sources were separated by SDS-PAGE. Proteins were transferred onto PVDF membranes (Mil-lipore) at 4 °C and then probed with primary antibodies over-night with shaking at 4 °C. The blots were washed with PBST(PBS plus 0.5% Tween 20) and incubated with the appropriateHRP-conjugated secondary antibodies. Immunoblots weredetected with an ECL Plus kit (Thermo Scientific/Pierce). Theantibodies used in this study were as follows: anti-type I colla-gen antibody (Novus Bio, Littleton, CO); anti-Smad2/3 andanti-p-Smad2 (Cell Signaling Technologies, Beverly, MA); anti-fibronectin, anti-desmin, and anti-�-SMA (Sigma-Aldrich),anti-myc antibody (clone 9E10, Life Technologies); and anti-GAPDH (Santa Cruz Biotechnology, Dallas, TX).

Plasmids and Cell Cultures—Human and mouse keratino-cytes were purchased from Zen-Bio Inc. (Research TrianglePark, NC) and cultured according to the manuals of the manu-facturer. 3T3 cells were obtained from the ATCC. Transfectionof GFP-MG53 into human keratinocytes was performed usingLipofectamine LTX reagent (Life Technologies) according tothe instructions of the manufacturer. 3T3 cells were main-tained in DMEM supplemented with 10% FBS and 1% peni-cillin/streptomycin. 3T3 cells were transfected with thepcDNA-myc-MG53 plasmid (23), and cells stably expressingmyc-MG53 protein were selected with G148 (Sigma) for 2weeks and confirmed by Western blotting with anti-MG53 andanti-myc antibodies. All cells were maintained in mediumunder 5% CO2 culture conditions. 3T3 cells were seeded andcultured in complete medium (DMEM and 10% FBS) in T25flasks for 16 h until 70% confluence. Cells were washed twicewith serum-free DMEM and then subjected to various treat-ments, including TGF-� (10 ng/ml), rhMG53 (60 �g/ml), or acombination of both TGF-� and rhMG53.

MTT Cell Viability Assay—To evaluate the effect of rhMG53on the proliferation of 3T3 cells, an 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was used.3T3 cells were plated at 1000 –1500 cells/well in a 96-well plateand then treated with indicated amounts of rhMG53 for 24 h.20 �l of MTT (5 mg/ml in PBS) was added to each well andincubated further for 4 h. Absorbance was recorded at 570 nmafter adding 150 �l of dimethyl sulfoxide.

In Vitro Cell Membrane Injury Assays—A suspension of pri-mary human keratinocytes (9 � 104 cells/150 �l of PBS/96-welldish) was added to the wells of a 96-well plate along with acid-washed glass microbeads and the indicated amount of rhMG53(0 –200 �g/ml). The plate was shaken at 200 rpm for 6 min toinduce cell membrane damage and then centrifuged at 3000 �g for 5 min to collect cells and microbeads at the bottoms of thewells. 50 �l of supernatant was transferred from the well to anew 96-well plate for a lactate dehydrogenase release assay witha lactate dehydrogenase cytotoxicity detection kit (Thermo Sci-entific). Measurement of the release of lactate dehydrogenasefollowing microglass bead damage was used as an index ofmembrane injury (18, 23). The released lactate dehydrogenasewas calculated by subtracting the basal lactate dehydrogenaserelease from wells without glass beads (no damage) from theexperimental conditions.

In Vitro Cell Migration Assay—3T3 cells or cells stablyexpressing myc-MG53 were grown to 90% confluence in com-plete medium in a 6-well plate. Cells were then switched to amedium containing rhMG53 (60 �g/ml) and subjected to ascratch wound assay with 200-�l pipette tips, following the pro-tocol described previously (24). Time-dependent cell migrationwas recorded using a Zeiss Axiovert 200 phase-contrast micro-scope 0, 6, and 22 h post-injury.

Encapsulation of rhMG53 in Hydrogel—Hydrogel was syn-thesized through free radical polymerization of N-isopropylac-rylamide, acrylic acid, and hydroxyethyl methacrylate-oligohy-droxybutyrate (Sigma) as described previously (25, 26). In brief,N-isopropylacrylamide, acrylic acid, and hydroxyethyl methac-rylate-oligohydroxybutyrate at a molar ratio of 86/4/10 weredissolved in dioxane in a three-necked flask. The solution wasbubbled in nitrogen gas for 15 min before the degassed benzylperoxide solution was injected. After 24 h of reaction at 70 °C,the solution was cooled to room temperature and precipitatedwith hexane. The polymer was purified twice using THF/di-ethyl ether, and the resulting polymer was vacuum-dried over-night. An equal volume of rhMG53 (2 mg/ml in PBS) was mixedthoroughly with hydrogel (15% (w/v) in PBS) solution at 4 °Covernight to encapsulate rhMG53 in hydrogen (rhMG53/hy-drogel) to a final concentration of 1 mg of rhMG53/1 ml ofhydrogel.

Immunocytochemistry—3T3 cells grown on glass coverslipswere subjected to treatments with the control (PBS), rhMG53,TGF-�, or a combination of both. Cells were then fixed with 4%paraformaldehyde for 30 min at room temperature, permeabi-lized (0.2% Triton X-100), blocked with BSA, and stained withprimary antibodies (anti-phospho-Smad2/3 (p-Smad2/3) oranti-�-SMA) overnight at 4 °C with rocking. Cells were thenincubated with fluorophore-conjugated secondary antibodies(Life Technologies) for 2 h at room temperature. F-actin wasvisualized using Alexa Fluor 647 phalloidin (Life Technologies).Nuclear staining was performed with DAPI (Sigma). Coverslipswere washed and mounted with Fluoromount G (ElectronMicroscopy Sciences, Hatfield, PA). Images were taken andanalyzed using a Zeiss 780 confocal microscope. At least 100stained cells/sample/experiment were analyzed.

Statistical Analysis—All data are expressed as mean � S.D.Groups were compared by Student’s t test and analysis of vari-

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ance for repeated measures. A value of p � 0.05 was consideredsignificant.

Results

Defective Skin Structure in mg53�/� Mice—To investigatethe biological function of MG53 in repairing skin injury, weused Western blot analysis and immunohistochemical stainingto determine the expression of MG53 in skin tissues (Fig. 1).Although MG53 was detected in mouse skin, keratinocytesderived either from human or mouse did not express MG53(Fig. 1a). Immunohistochemical staining confirmed that MG53is localized to the panniculus carnosus, a subdermal striatedmuscle layer present in many rodents (Fig. 1b). This result isconsistent with our previous observation that MG53 is pre-dominantly expressed in striated muscle tissues (14, 27).

Despite the absence of MG53 expression in keratinocytes,remarkable defects in skin architecture were observed inmg53�/� mice. Masson trichrome staining revealed elevatedcollagen deposition in the mg53�/� dermal layer comparedwith their WT littermates (Fig. 1c). Measurement of thehydroxyproline content in the skin tissue indicated thatmg53�/� skin contained �40% more collagen than WT skin(Fig. 1d). In addition, mg53�/� skin possessed roughly 30%fewer hair follicles than WT littermates (Fig. 1e).

Although MG53 is absent from keratinocytes and fibroblasts,circulating MG53 may contribute to the maintenance of nor-mal skin architecture under physiological conditions. Our pre-vious studies have shown that MG53 present in the bloodstream plays a protective role against tissue injuries (18 –21).The present data show that genetic ablation of MG53 leads toabnormal skin architecture, hair follicle formation, and colla-gen overproduction in the dermal layer.

Knockout of MG53 Impairs Wound Healing and IncreasesScar Formation—Given the abnormal skin phenotype ofmg53�/� mice, we evaluated the role of MG53 in wound heal-ing using an established excisional cutaneous wounding model(28 –30). Wounds were created in the dorsal region of WT andmg53�/� animals using a biopsy punch to cut through both theepidermal and dermal layers (Fig. 2a). Wound size was thenmonitored over the subsequent 15 days. In WT mice, woundretraction occurred within the first few days and was signifi-cantly greater on day 1 compared with mg53�/� mice. Delayedwound healing in mg53�/� mice was observed consistently onday 7 and beyond (Fig. 2b). On day 11 post-injury, Massontrichrome staining of mg53�/� skin revealed excessive collagendeposition in the interstitial area of the injured muscle fibers(Fig. 2c, bottom panel). The extent of interstitial collagen dep-osition was significantly lower in WT littermates (Fig. 2c, toppanel). Quantification of collagen deposition in WT andmg53�/�mice is summarized in Fig. 2d.

MG53 Protects against Keratinocyte Injury—Primary cul-tured human keratinocytes were transfected with GFP-MG53,a fusion construct with GFP added to the amino-terminal endof MG53, to investigate the extent of MG53 participation in therepair of membrane injury. As shown in Fig. 3a, right panel, inresponse to injury caused by penetration of a microelectrodeinto the plasma membrane, GFP-MG53 rapidly translocatedtoward the acute injury site. This GFP-MG53 translocation to

FIGURE 1. MG53 knockout mice show abnormal skin structure. a, Westernblot analysis of MG53 was performed with mouse skin lysates (lane 1, 40 �g),mouse and human keratinocytes (lanes 2 and 3, 40 �g), and skeletal musclederived from mg53�/� mice (�/�, lane 4, 10 �g) and WT mice (�/�, lane 5, 10�g). Desmin was used as a marker for skeletal muscle, and tubulin was used asa protein loading control. b, immunohistochemical staining illustrating thepresence of MG53 in the panniculus carnosus, a subdermal muscle layer, inWT mice (left panel) but absent in mg53�/� mice (right panel). c, Massontrichrome staining revealed elevated collagen deposition in mg53�/� mouseskin (bottom panel) compared with WT skin (top panel). d, quantification ofhydroxyproline content showed higher collagen content in mg53�/� skincompared with WT skin. *, p � 0.05 (n � 6). e, quantification of the number ofhair follicles in mouse skin. **, p � 0.01 (n � 12).

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membrane injury sites in keratinocytes is similar to thatobserved in C2C12, HEK293, and other cell types (14, 18).

We have shown previously that rhMG53 protein could pro-tect various cell types against cell membrane disruption whenapplied to the extracellular environment (18). To quantify theeffect of rhMG53 protection against injury to keratinocytes, wefirst employed an in vitro keratinocyte cell membrane damageassay. In this assay, we measured the amount of lactate dehy-drogenase leaked from the cell interior into the extracellularsolution following cell membrane injury induced by microglassbeads (18, 23). As shown in Fig. 3b, application of rhMG53 to

the extracellular solution reduced the release of lactate dehy-drogenase from keratinocytes into the culture medium in adose-dependent manner after microglass bead treatment. Thissuggests that rhMG53 protects against mechanical injury tokeratinocytes.

MG53 Enhances Fibroblast Migration following ScratchWounding—The wound healing process involves migration offibroblasts to wound sites and differentiation of fibroblasts intomyofibroblasts (2, 4, 5, 8, 11). To test whether MG53 plays a rolein the modulation of fibroblast migration during wound heal-ing, we performed an in vitro scratch wound assay with cultured3T3 fibroblast cells (Fig. 4a). 3T3 fibroblasts grown to �95%confluence were subjected to scratch wounding. The cells weretreated with phosphate-buffered saline (Fig. 4a, left column),rhMG53 (60 �g/ml, center column), or stably transfected withmyc-MG53 (right column). Images were captured 0, 6, and 22 hpost-injury. Cell migration was quantified by the distance trav-eled by cells from the scratch edge toward the center of thescratch (Fig. 4b). Both rhMG53 treatment and myc-MG53overexpression enhanced the migration of fibroblast cells fol-lowing scratch wounding.

The MG53-mediated enhancement of fibroblast migrationwas not due to an increase in cell proliferation, as determinedby MTT assay because the cell growth rate was not affected bythe treatment with rhMG53 (Fig. 4c). Under confocal micros-copy, we found that fibroblasts cultured in serum-free mediumdisplayed a formation of intracellular stress fibers that were

FIGURE 2. mg53�/� mice display delayed wound healing and excessivecollagen deposition following injury. a, representative images of cutane-ous wounds from WT (top panel) and mg53�/� (bottom panel) mice at differ-ent time points. D, day. b, statistical analyses revealed that mg53�/� micedisplayed delayed wound healing compared with their WT littermates. **, p �0.01 (n � 12). c, Masson trichrome staining of wound sections derived fromWT (top panel) and mg53�/� mice (bottom panel) 10 days post-injury. Arrowsindicate fibrosis and deposition of collagen. d, quantification of collagen dep-osition in dorsal skin on day 10 following excisional wounding in WT andmg53�/� mice. **, p � 0.01 (n � 6/group).

FIGURE 3. MG53 protects human keratinocyte from membrane injury. a,GFP-MG53 expressed in human keratinocytes rapidly translocated to theacute cell membrane injury site created by penetration of a microelectrode(arrow). A live-cell imaging movie of the cell response to injury can be found insupplemental Movie 1. b, rhMG53 protected human keratinocytes from glassmicrobead-induced membrane damage in a dose-dependent manner (n � 3independent experiments). LDH, lactate dehydrogenase.

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larger and more contiguous compared with those treated withrhMG53 (these were thinner and dissociated) (Fig. 4d). Otherinvestigators have shown that reduced stress fiber formation iscorrelated with the enhanced cell migration associated withwound healing (31, 32). Together, these results show thatMG53-mediated reduction of stress fiber formation may be acontributing factor for the enhancement of cell migration dur-ing wound healing.

Subcutaneous Administration of rhMG53 accelerates WoundHealing—We have shown previously that systemic administra-tion of rhMG53 provided dose-dependent protection againstinjury to muscles and the heart, kidneys, and lungs under vari-

ous injurious conditions (18 –21). We studied the effect ofrhMG53 in protection against dermal injury using establishedrat models of wound healing (29, 30, 33). Excisional woundswere created on the back of rats with a biopsy punch (diameter,6 mm) (Fig. 5a). Vehicle or rhMG53 (0.2 mg/wound) wasapplied to the wound site via subcutaneous injection afterinjury, and on the following 3 days (once per day). We foundthat wounds treated with rhMG53 displayed better healingcompared with those treated with vehicle (Fig. 5b). On day 1,rhMG53-treated wounds showed improved settlement in thewound bed compared with vehicle-treated wounds despitethere being no significant difference in wound closure between

FIGURE 4. rhMG53 enhances fibroblast migration following scratchwounding. a, monolayers of 3T3 fibroblasts were subjected to scratchwounding. The cells were treated with vehicle (PBS, left column), rhMG53(center column), or 3T3 cells stably expressing myc-MG53 (right column) inDMEM. Representative images were captured 0, 6, and 22 h post-injury. b,both rhMG53 treatment and myc-MG53 overexpression enhanced the migra-tion of fibroblast cells following scratch wounding (n � 3 independent assaysfor all groups). **, p � 0.01). c, 3T3 cells were treated with different concen-trations (0 – 60 �g/ml) of rhMG53 for 24 h, and cell growth and viability wereanalyzed by MTT assay (data represented three independent assays). d, sub-confluent 3T3 cells were grown in serum-free medium in the absence ofrhMG53 (PBS, left panel) or in the presence of rhMG53 (60 �g/ml, right panel)for 48 h. Cells were fixed and stained with Alexa Fluor 647 phalloidin.

FIGURE 5. rhMG53 improves dermal wound healing in rats. a, representa-tive micrograph images of rat excisional wounds were taken at different timepoints. Wounds were created on the back skin of rats and treated with saline(top row) or rhMG53 (0.2 mg/wound, subcutaneous injection, bottom row)and three additional daily treatments. D, day. b, quantification of wound heal-ing of rhMG53-treated rats compared with saline-treated control rats. **, p �0.01 (n � 8/group). c, incisional wound images at different time points in ratsreceiving saline as a control (top row) or a subcutaneous injection of rhMG53(bottom row). d, quantification of incisional wound healing in rats treated withrhMG53 compared with controls. **, p � 0.01 (n � 8/group).

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the two groups. Improved wound closure was clearly observedstarting on day 5 in the rhMG53-treated group. The role ofrhMG53 in facilitating wound healing was tested further in anincisional wound model. Consistent with our observations inthe excisional wound model, rhMG53 treatment led to signifi-cant improvement in healing of the incisional wound startingon day 3 and continuing to wound closure (Fig. 5, c and d).These studies suggest that subcutaneous delivery of rhMG53has beneficial effects on wound healing.

Hydrogel Encapsulation Provides Efficient Delivery ofrhMG53 to Dermal Wounds—Pharmacokinetic studies haveshown that rhMG53 has a short half-life of �1.5 h in the bloodcirculation after intravenous or subcutaneous administration(18, 21). Therefore, treatment of open dermal wounds requiredrepetitive subcutaneous administration of rhMG53. Our grouphas previously developed a thermosensitive hydrogel drugdelivery system that is biodegradable and safe for topical appli-cation (25). rhMG53 encapsulated in hydrogel exists in liquidgel format at room temperature and quickly solidifies to form aflexible solid gel when at 37 °C (body temperature). This hydro-gel encapsulation allows better retention and time-controlledrelease of rhMG53 at the wound site to facilitate wound healing.

We investigated the therapeutic efficacy of the rhMG53/hy-drogel formulation in the rat incisional wound model. Asshown in Fig. 6a, rat skin incisions that received a local admin-istration of rhMG53/hydrogel (0.2 mg rhMG53/wound) healedsignificantly faster than those treated with hydrogel alone. Sta-tistical analyses with multiple animals indicate that rhMG53treatment both improves wound healing (Fig. 6b) and reducesscar formation (Fig. 6c). On day 18 post-injury, scars wereclearly diminished in rats treated with hydrogel/rhMG53 com-pared with animals treated with hydrogel alone (Fig. 6c, leftpanels). Histological analysis was used to examine collagendeposition in sections of gastrocnemius muscle by Massontrichrome staining (Fig. 6c, right panels). The results showedthat rhMG53/hydrogel treatment markedly reduced collagencontent (blue staining) on day 8 post-injury (summarized in Fig.6d). Similar results have been reported in our previous studies,where systemic application of rhMG53 reduced the fibroticcontent in skeletal (18) and cardiac muscle (20).

To further assess the effect of rhMG53 in reducing fibrosisduring wound healing, we used a rabbit model of dermal inci-sional wounding. An incisional wound 2 cm in length and 0.5cm in depth was created on the back of the rabbit (Fig. 6e).Animals received either hydrogel or rhMG53/hydrogel on thewound sites immediately after injury, followed by anotheradministration on day 1 post-wounding. Rabbits treated withrhMG53/hydrogel healed better than those receiving hydrogelalone. Masson trichrome staining of the skeletal muscle layerwas conducted 14 days post injury. Sections derived from rab-bits receiving rhMG53/hydrogel treatment showed signifi-cantly less fibrosis compared with controls (Fig. 6f). Together,our in vivo studies with mouse, rat, and rabbit models of dermalinjury support the therapeutic effect of rhMG53 in facilitatingwound healing and inhibiting scar formation.

MG53 Modulates Fibroblast-to-Myofibroblast Differentia-tion to Reduce �-SMA Expression—TGF-� is a growth factorthat regulates wound healing and fibrotic remodeling associ-

ated with the healing process (7, 34 –36). Many studies havesuggested that TGF-� contributes to wound healing and scar-ring by controlling the differentiation of myofibroblasts fromfibroblasts (5, 8, 34). The expression of �-SMA is considered amarker of myofibroblasts differentiation (8, 35). We thereforeexamined whether MG53 affects the expression level of �-SMAassociated with fibroblast differentiation. Western blottingshowed an elevation of �-SMA in skin tissues derived frommg53�/� mice compared with WT mice (Fig. 7, a and b), sug-gesting the possibility that MG53 in the circulation may con-tribute to fibroblast differentiation.

Changes in the expression of �-SMA were assessed in cul-tured 3T3 fibroblasts with or without rhMG53 (60 �g/ml) orTGF-� (10 ng/ml) treatment a or combination of TGF-� andrhMG53 in serum-free medium for 4, 48, or 72 h (Fig. 7c).Although TGF-� stimulation led to a more than 3-fold increasein the expression of �-SMA, rhMG53 significantly suppressedTGF-� induced �-SMA expression in 3T3 cells (Fig. 7d). Im-munofluorescence staining of �-SMA and phalloidin revealedthe diminished appearance of F-actin stress fibers in cellstreated with both TGF-� and rhMG53 (Fig. 7e), suggestingthat rhMG53 treatment prevents the differentiation ofmyofibroblasts.

MG53 Affects TGF-� Signaling to Control Fibrotic Remodel-ing during Wound Healing—Many studies have suggested thatthe TGF-�/Smad axis is a principal signaling pathway for con-trolling the transcriptional activation of �-SMA, fibroblast-myofibroblast differentiation, and secretion of ECM proteinsduring the fibrogenic response of wound healing (8, 10, 37–39).This TGF-�-mediated signaling involves the nuclear transloca-tion of p-Smad2/3 to stimulate the expression of downstreamsubstrates and the ECM proteins, such as fibronectin and col-lagen 1. We examined the activation of Smad2/3 signaling withbiochemical and immunocytochemical staining approaches on3T3 cells undergoing similar treatments as described above.Although TGF-�-dependent activation of p-Smad2/3 wasobserved in 3T3 cells, cells treated with both rhMG53 andTGF-� clearly showed a reduced activation of p-Smad2/3 (Fig.8, a and b).

Taking advantage of the monoclonal antibody that recog-nizes the phosphorylated form of Smad2/3, we examined thenuclear translocation of Smad2/3 in 3T3 cells following stimu-lation with TGF-� with or without cotreatment of rhMG53(Fig. 8, c and d). Localization of Smad2/3 in the cell nuclei wasobserved infrequently in 3T3 cells maintained under basalserum deprivation conditions (Fig. 8c). Treatment with TGF-�(10 ng/ml) for 4 h led to a significant increase in nuclear local-ization of Smad2/3 because more than 90% of 3T3 cells werepositive for immunofluorescence labeling with p-Smad2/3.However, cotreatment of rhMG53 diminished the TGF-�-in-duced nuclear localization of Smad2/3 because only 68% of cellswere observed with pSmad2/3 nuclear translocation (Fig. 8d).

We next investigated the expression levels of ECM substratessuch as collagen type 1 and fibronectin (FN). We found thattreatment of 3T3 cells with rhMG53 led to a significant reduc-tion in FN under serum-deprived conditions (Fig. 8e). Althoughthe level of FN was increased significantly following stimulationwith TGF-�, TGF-�-mediated elevation of FN expression was

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largely suppressed following cotreatment with rhMG53. Simi-larly, the expression of collagen type 1 from cells receivingcotreatment of TGF-� and rhMG53 showed a significantlylower expression compared with those receiving TGF-� treat-ment alone (Fig. 8f).

Discussion

Our group has demonstrated previously that MG53 acts asan essential component in the cell membrane repair machinery(14) and that the rhMG53 protein can protect against injury invarious cell and tissue types when applied to the extracellularenvironment or administered systemically (18 –21). In this

study, we show that MG53 has therapeutic benefits in treatingdermal wounds. We provide evidence that topical administra-tion of rhMG53 not only facilitates wound healing but also sup-presses scar formation. These studies reveal a role for MG53 inthe modulation of TGF-�-dependent fibroblast-myofibroblastdifferentiation in addition to its conventional cell membranerepair function.

Because MG53 is predominantly expressed in striated mus-cle tissues, most published studies have focused on character-izing the skeletal muscle and cardiac phenotype of mg53�/�

mice (14, 16). Here we show that, despite the absence of MG53expression in keratinocytes and fibroblasts, genetic ablation of

FIGURE 6. Hydrogel delivery of rhMG53 enhances wound healing and reduces scar formation. a, incisional wounds in the hind limbs of rats were treatedwith hydrogel (top row) or hydrogel encapsulating rhMG53 (bottom row). 200 �l of the hydrogel or rhMG53/hydrogel (1 mg/ml) was applied immediately afterwounding (0 h, n � 8/group). b, time-dependent wound closure compared with the original wound sizes. Hydrogel/rhMG53 treatment enhanced healing ofthe incisional wound. **, p � 0.01 (n � 8/group). c, representative macroscopic (left panels) and microscopic (right panels) images of skin treated with hydrogel(top panels) or rhMG53/hydrogel (bottom panels). The macroscopic images were taken from day 18 post-injury to observe scar formation (left panels). Massontrichrome staining of gastrocnemius muscle was performed on day 8 post-injury (right panels). Insets, decreased collagen deposition in muscle sectionsreceiving rhMG53/hydrogel treatment (bottom panels) compared with those receiving hydrogel alone (top panels). d, quantification of collagen deposition ofthe gastrocnemius muscle from animals treated with hydrogel or rhMG53/hydrogel on day 8 after incisional wounding. **, p � 0.01 (n � 8/group). e,macroscopic images of wounds in rabbits were taken from day 14 after the incisional wound injuries. f, Masson trichrome staining of rabbit skeletal muscletaken from the wound sites as indicated above on day 14 post-injury. Muscle sections derived from the rabbit receiving rhMG53/hydrogel treatment show lessfibrosis (collagen deposition, blue) compared with those of the control.

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MG53 leads to prominent defects in skin structure and itswound healing property. Excessive collagen deposition in thedermal layer of mg53�/� mice may reveal a compensatoryresponse of the animal to the chronic absence of MG53 in theblood circulation. The delayed wound healing response ofmg53�/� skin further suggests that MG53 in circulation mayfunction to protect the dermal layer under injurious conditions.

The therapeutic effect of rhMG53 on the facilitation ofwound healing was evaluated in three animal models, includingincisional and excisional wounds in rats and incisional woundsin rabbits. Although subcutaneous delivery of rhMG53 waseffective in accelerating skin wound healing, the short half-lifeof rhMG53 in the circulation necessitated repeated administra-tion (18 –21). We encapsulated rhMG53 in a thermosensitive

hydrogel to control its release at the wound site. The thermo-sensitive hydrogel can be mixed readily with rhMG53 in liquidformulation at 4 °C to room temperature, facilitating conven-ient topical administration. Upon delivery to the wound siteat body temperature (�37 °C), rhMG53/hydrogel solidifiesquickly, allowing the retention of rhMG53 at the injury site.This approach enhanced the effective dosage of rhMG53 at thewound site, achieving improved therapeutic efficacy of the pro-tein in wound healing compared with the conventionalapproach of repeated subcutaneous delivery. Hydrogel-medi-ated delivery of rhMG53 represents a potentially novel andeffective means to treat dermal injuries.

Through these studies, we identified the exciting phenome-non that rhMG53 suppresses scar formation during wound

FIGURE 7. rhMG53 treatment suppresses TGF�-mediated �-SMA activa-tion in fibroblasts. a, Western blotting shows elevated levels of �-SMA inskin tissues derived from mg53�/� mice compared with those from WT litter-mates. GAPDH was used as a loading control. b, summary data from multipleanimals. **, p � 0.01. (n � 6/group). c, immunoblots of �-SMA expression incultured 3T3 fibroblasts grown in serum-free medium in the presence ofTGF-� (10 ng/ml), rhMG53 (60 �g/ml), or both TGF-� and rhMG53. Westernblotting shows that rhMG53 suppressed TGF-�-mediated activation of�-SMA, with a maximal effect at the 72-h time point. d, the relative expressionof �-SMA at different time points. Data are the average of three independentexperiments. **, p � 0.01. e, immunofluorescence staining of �-SMA (green)and F-actin (phalloidin staining, red) reveals the reduction of stress fibers incells treated with TGF-� plus rhMG53 (right panel) compared with cellstreated with TGF-� alone (left panel) (n � 3 independent experiments).

FIGURE 8. rhMG53 negatively regulates TGF-�/Smad-dependent signal-ing and suppresses the synthesis of ECM proteins. a, immunoblots ofMG53, p-Smad2/3, total Smad 2/3, and GAPDH expression were derived from3T3 cells treated for 4 h under various culture conditions as denoted at the topof the blots. b, data represent the relative level of p-Smad2/3 as normalized tothe expression of total Smad2/3. c, immunofluorescence staining of �-SMA(green), p-Smad2/3 (red), and nuclei (DAPI, blue) derived from 48-h cultures of3T3 cells under the indicated conditions. Cells were treated with serum-freemedium (control), rhMG53 (60 �g/ml), TGF-� (10 ng/ml), or both. d, quantifi-cation of p-Smad2 nuclear translocation (n 120 cells for each of the threeindependent experiments). **, p � 0.01 for comparison between the indi-cated groups. e and f, rhMG53 suppresses TGF�-mediated expression of FN(e) and collagen 1 (Col-1) in 3T3 cells treated for 48 h. (f). The relative expres-sion of collagen-1 and fibronectin was calculated from four independentexperiments. **, p � 0.01.

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healing. The role of MG53 in the modulation of scarring wasfirst observed in mg53�/� mice. These animals displayed exces-sive fibrotic remodeling following dermal injury. With hydro-gel-mediated delivery of rhMG53, reduced scar formation wasobserved consistently in both rat and rabbit models of dermalinjuries. The promotion of scarless wound healing is a majorchallenge because the natural biological wound healing processis often accompanied by fibrotic remodeling because of the lim-ited regenerative capacity of the dermis. Our finding thatrhMG53 facilitates wound healing and reduces scarring haspotentially broad applications in treating tissue injuries whereexcessive scarring can be detrimental to the biological functionof the organ.

We conducted biochemical and cellular studies to under-stand the mechanisms that underlie the biological function ofMG53 in facilitating wound healing and suppressing scar for-mation (Fig. 9). First, MG53, as a membrane repair molecule,can protect against acute injuries to keratinocytes at the dermalinjury site. Such a fast membrane resealing response couldreduce the level of inflammatory cytokines released to thewound milieu, significantly affecting immunogenic and fibroticremodeling during later stages of healing. Second, MG53 pres-ent in the extracellular solution can facilitate the migration offibroblast cells in response to dermal wounding, which is animportant step in efficient wound healing. Confocal micro-scopic imaging revealed that fibroblast cells exposed torhMG53 show reduced formation of stress fibers, which canlead to increased mobility of fibroblasts during wound healing.Third, MG53 can interfere with TGF-� signaling to control thedifferentiation process of fibroblasts into myofibroblasts. The

latter is a major source of secretion of ECM proteins and themain factor contributing to scarring. We have data showingthat treatment of fibroblasts with rhMG53 leads to reducedexpression of �-SMA and ECM components, which underliereduced scar formation during wound healing.

Fibroblasts play important roles in wound repair, and theiruncontrolled differentiation into myofibroblasts is responsiblefor the excessive synthesis of extracellular matrix proteins aspart of scar formation (8, 11, 39 – 41). TGF-� is an importantcytokine that controls the conversion of fibroblasts into myofi-broblasts. Although many studies have been designed to targetTGF-� signaling for the treatment of wound healing, theseattempts often faced a double-edged sword. For example,blocking TGF-� signaling can reduce scarring but can alsocompromise wound healing and other immune functions (5,10, 39, 40, 42). Our identification of a functional link betweenMG53 and TGF-� signaling has important therapeutic impli-cations regarding wound healing. The trifunctional role ofMG53 in wound healing, e.g. protection against acute injury tocell membrane, promotion of fibroblast migration, and controlof myofibroblast differentiation, has advantages over many ofthe current approaches for the treatment of wound healing.Although the biological function of MG53 in cell membranerepair has received much attention, the cytokine function ofMG53 in controlling fibroblast-myofibroblast differentiationopens a new avenue for reducing fibrotic remodeling inresponse to recovery from traumatic injuries not only to thedermal layer but also to many vital organs, such as myocardialinfarction, acute lung injury, and acute kidney injury, whereprevention of scarring is of prominent importance for restoring

FIGURE 9. Schematic illustrating the role of MG53 in wound healing and scar formation. a, MG53 nucleates the cell membrane repair machinery andprotects against acute injury to keratinocytes and fibroblasts in response to dermal injury. b, MG53 promotes the migration of fibroblasts to the wound sites bymodulating stress fiber formation. c, MG53 regulates TGF-�/Smad signaling to control the differentiation of fibroblasts into myofibroblasts, leading todown-regulation of ECM proteins during the tissue remodeling process of wound healing.

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normal organ function (18 –21). One avenue for future studymay include elucidating the molecular interaction betweenMG53 and TGF-� signaling cascades, information that will beinstrumental for developing effective therapies to facilitatescarless wound healing.

Author Contributions—H. L. and J. M. developed the concept for thestudy. H. L., P. D., D. H., H. Z., S. M. S., E. J., and C. Z. performed theanimal model studies with wound healing. H. L., P. L., L. Z., X. Z.,M. S., M. F., M. O., and M. S. conducted biochemical studies, the invitro cell assay, and imaging. Z. F. and J. G. performed hydrogel stud-ies. T. T. and H. M. performed toxicological studies of rhMG53. J. M.oversaw the entire project. H. L., S. S., R. H., J. G., and J. M. wrote themanuscript.

Acknowledgments—We thank Novoprotein Scientific, Inc., for therhMG53 protein.

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Zeng, Jianjun Guan and Jianjie MaDuofen He, Steven M. Steinberg, Robert Higgins, Hua Zhu, Elizabeth John, ChunyuMingzhai Sun, Minghuan Fu, Matthew Orange, Matthew Sermersheim, Hanley Ma, Haichang Li, Pu Duann, Pei-Hui Lin, Li Zhao, Zhaobo Fan, Tao Tan, Xinyu Zhou,

Cell Membrane RepairModulation of Wound Healing and Scar Formation by MG53 Protein-mediated

doi: 10.1074/jbc.M115.680074 originally published online August 25, 20152015, 290:24592-24603.J. Biol. Chem. 

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